mitoBKCa Channel Research Articles

The influence of flavonoids on oxygen consumption and membrane potential of isolated endothelial mitochondria via modulation of mitoBKCa channel activity

The influence of flavonoids on oxygen consumption and membrane potential of isolated endothelial mitochondria via modulation of mitoBKCa channel activity

Metal ion-substituted protoporphyrins as a tool to study regulation of the activity of mitoBKCa channel by hydrogen sulfide

Metal ion-substituted protoporphyrins as a tool to study regulation of the activity of mitoBKCa channel by hydrogen sulfide

A concise discussion of the regulatory role of cGMP kinase I in cardiac physiology and pathology.

The underlying cause of cardiac hypertrophy, fibrosis, and heart failure has been investigated in great detail using different mouse models. These studies indicated that cGMP and cGMP-dependent protein kinase type I (cGKI) may ameliorate these negative phenotypes in the adult heart. Recently, evidence has been published that cardiac mitochondrial BKCa channels are a target for cGKI and that activation of mitoBKCa channels may cause some of the positive effects of conditioning in ischemia/reperfusion injury. It will be pointed out that most studies could not present convincing evidence that it is the cGMP level and the activity cGKI in specific cardiac cells that reduces hypertrophy or heart failure. However, anti-fibrotic compounds stimulating nitric oxide-sensitive guanylyl cyclase may be an upcoming therapy for abnormal cardiac remodeling.

Mechanosensitivity of mitochondrial large-conductance calcium-activated potassium channels

Potassium channels have been discovered in the inner mitochondrial membrane of various cells. These channels can regulate the mitochondrial membrane potential, the matrix volume, respiration and reactive species generation. Therefore, it is believed that their activation is cytoprotective in various tissues. In our study, the single-channel activity of a large-conductance calcium-activated potassium channel (mitoBKCa) was measured by the patch-clamp technique on mitoplasts derived from mitochondria isolated from human glioma U-87 MG cells. Here, we show for the first time that mechanical stimulation of mitoBKCa channels results in an increased probability of channel opening. However, the mechanosensitivity of mitoBKCa channels was variable with some channels exhibiting no mechanosensitivity. We detected the expression of mechanosensitive BKCa-STREX exon in U-87 MG cells and hypotesize, based on previous studies demonstrating the presence of multiple BKCa splice variants that variable mechanosensitivity of mitoBKCa could be the result of the presence of diverse BKCa isoforms in mitochondria of U-87 MG cells. Our findings indicate the possible involvement of the mitoBKCa channel in mitochondria activities in which changes in membrane tension and shape play a crucial role, such as fusion/fission and cristae remodeling.

Mitochondrial BK Channel Openers CGS7181 and CGS7184 Exhibit Cytotoxic Properties.

Potassium channel openers (KCOs) have been shown to play a role in cytoprotection through the activation of mitochondrial potassium channels. Recently, in several reports, a number of data has been described as off-target actions for KCOs. In the present study, we investigated the effects of BKCa channel openers CGS7181, CGS7184, NS1619, and NS004 in neuronal cells. For the purpose of this research, we used a rat brain, the mouse hippocampal HT22 cells, and the human astrocytoma U-87 MG cell line. We showed that CGS7184 activated the mitochondrial BKCa (mitoBKCa) channel in single-channel recordings performed on astrocytoma mitoplasts. Moreover, when applied to the rat brain homogenate or isolated rat brain mitochondria, CGS7184 increased the oxygen consumption rate, and can thus be considered a potentially cytoprotective agent. However, experiments on intact neuronal HT22 cells revealed that both CGS7181 and CGS7184 induced HT22 cell death in a concentration- and time-dependent manner. By contrast, we did not observe cell death when NS1619 or NS004 was applied. CGS7184 toxicity was not abolished by BKCa channel inhibitors, suggesting that the observed effects were independent of a BKCa-type channel activity. CGS7184 treatment resulted in an increase of cytoplasmic Ca2+ concentration that likely involved efflux from internal calcium stores and the activation of calpains (calcium-dependent proteases). The cytotoxic effect of the channel opener was partially reversed by a calpain inhibitor. Our data show that KCOs under study not only activate mitoBKCa channels from brain tissue, but also induce cell death when used in cellular models.

Identification of Large-Conductance Calcium-Regulated K Channel in Human Dermal Mitochndria

Potassium channels have been found in the inner mitochondrial membrane of various cells. These channels regulate the mitochondrial membrane potential, respiration and the production of reactive oxygen species. In this study, we identified the activity of a large-conductance Ca2+-regulated potassium channel (mitoBKCa channel) in mitoplasts isolated from a primary human dermal fibroblast cell line. A potassium selective current was recorded with a mean conductance of 280 ± 2 pS in a symmetrical 150 mM KCl solution. The mitoBKCa channel was activated by the Ca2+ and by potassium channel opener NS1619. The channel activity was irreversibly inhibited by paxilline, a selective inhibitor of the BKCa channels. In isolated fibroblast mitochondria NS1619 depolarized the mitochondrial membrane potential, stimulated non-phosphorylating respiration and decreased superoxide formation. Additionally, the α and β subunits (predominantly in the β3 form) of the BKCa channels were identified in fibroblast mitochondria. Our findings indicate, for the first time, the presence of a large-conductance Ca2+-regulated potassium channel in the inner mitochondrial membrane of human dermal fibroblasts. We show that a large-conductance Ca2+-regulated potassium channel functions in the inner mitochondrial membrane of human dermal fibroblasts. The electrophysiological and biochemical properties of this channel is similar to previously described mitochondrial potassium channels from other cell types.This study was supported by a grant MERIS PBS1/B8/1/2012 from the National Centre of Research and Development, Poland and partially by grant 2015/17/B/NZ1/02496 from Polish National Science Center.

Flavonoids as Natural Modulators of Mitochondrial Potassium Channel

Flavonoids belong to a vast group of polyphenolic compounds that are widely distributed in all foods of plant origin. They exhibit a large variety of biological properties. Interest in health benefits of flavonoids has increased due to their potent antioxidant and free-radical scavenging activities. The biological activity, bioavailability and low toxicity set broad prospects of the usage of some of these substances as potential therapeutics for a number of human diseases. Some flavonoids have also been shown to be cardioprotective. Although the antioxidant effect of flavonoids has been long thought to be a crucial factor accounting for cellular cardioprotection, mitochondrial pathways (including mitochondrial ion channels) are presently emerging potential targets for a specific pharmacological action of some flavonoids in the anti-ischemic strategies.The detailed research describing a direct interaction of flavonoids with mitochondrial potassium channels is lacking. Therefore, the scientific aim of the these studies is the description of the regulation of the mitoBKCa and mitoKATP channels by selected flavonoids and functional consequences of this regulation.Our studies with mitoplasts isolated from primary human dermal fibroblast cells suggest that application of 10 μM naringenin leads to an increase in mitoBKCa channel activity. Statistical analysis of these results showed that opening probability of the channel decreased from 0.95 in the control conditions (100 μM Ca2+) to 0.06 at low calcium level (1 μM Ca2+) and increased to 0.81 after application of naringenin. Also, it has been observed that activity of the mitoKATP channel increases after application of 10 µM naringenin.This study was supported by a grant MERIS PBS1/B8/1/2012 from the National Centre of Research and Development, Poland and partially by The Nencki Institute of Experimental Biology, Poland.

A large-conductance calcium-regulated K+ channel in human dermal fibroblast mitochondria.

Potassium channels have been found in the inner mitochondrial membrane of various cells. These channels regulate the mitochondrial membrane potential, respiration and production of reactive oxygen species. In the present study, we identified the activity of a mitochondrial large-conductance Ca2+-regulated potassium channel (mitoBKCa channel) in mitoplasts isolated from a primary human dermal fibroblast cell line. A potassium selective current was recorded with a mean conductance of 280 ± 2 pS in a symmetrical 150 mM KCl solution. The mitoBKCa channel was activated by the Ca2+ and by potassium channel opener NS1619. The channel activity was irreversibly inhibited by paxilline, a selective inhibitor of the BKCa channels. In isolated fibroblast mitochondria NS1619 depolarized the mitochondrial membrane potential, stimulated nonphosphorylating respiration and decreased superoxide formation. Additionally, the α- and β-subunits (predominantly the β3-form) of the BKCa channels were identified in fibroblast mitochondria. Our findings indicate, for the first time, the presence of a large-conductance Ca2+-regulated potassium channel in the inner mitochondrial membrane of human dermal fibroblasts.

BK Channel Subproteome Includes Novel Mitochondrial Partners: ADP/ATP Carrier and TOM 22 Import Receptor

BK channels are large conductance voltage- and Ca2+-sensitive potassium-selective channels. A specific splice variant, BK-DEC (mitoBKCa), is targeted to mitochondria of adult cardiomyocytes. MitoBKCa activation protects the heart from ischemia and reperfusion injury. To understand its physiological relevance in the heart we characterized the mitoBKCa-associated subproteome in rat cardiomyocytes.Three strategies were implemented: 1) Pulldown of DEC-interacting cellular proteins from left ventriculocytes using recombinant GST-DEC 2) Pulldown of DEC-interacting mitochondrial proteins from isolated left ventriculocytes mitochondria using recombinant GST-DEC and 3) Immunoprecipitation of mitoBKCa-interacting mitochondrial proteins with BK monoclonal antibody and control IgG; mitochondrial proteins were purified from left ventricles. Protein composition was analyzed with 1-D-SDS-PAGE, followed by in-gel trypsinization and LC/MS/MS Mass Spectrometry. These approaches provide a detailed view of mitoBKCa associated subproteome in cardiomyocytes.We identified 516 interacting partners with mitoBKCa with a score>21, including 161 mitochondrial proteins. Gene ontology analysis of mitoBKCa-associated mitochondrial subproteome revealed mitoBKCa association with 13 distinct mitochondrial functions in the heart. For example, DEC interacts with ADP/ATP translocase 1 (AAC) (2 peptides, n=1, score=43), suggesting a role in energy balance. Additionally, DEC also associates with TOM22 (1 peptide, n=1, score=45), suggesting a BK mitochondrial import mechanism through the TOM system. We verified interactions of mitoBKCa with TOM22 and AAC by expressing BK and TOM22 or AAC in HEK and detecting their interactions with reciprocal coimmunoprecipitation. Second, we demonstrated that BK channel, TOM22 and AAC are targeted into mitochondria using cell fractionation and confocal microscope imaging. In conclusion, proteomic analysis identified a wide variety of cellular and mitochondrial proteins forming complex with mitoBKCa channels. BK association with AAC and TOM22 suggests its involvement in regulating mitochondrial energetic balance, and a TOM22 mediated mechanism for mitochondrial targeting.

Mouse Cardiac MitobkCa Associates with β1 Subunit Favoring Channel Expression and Activity

We previously showed that in rat cardiomyocytes two types of mitoBKCa are present with high and low Po (∼0.85 and ∼0.01, respectively, at −60 mV in 12 µM Ca2+). We hypothesized that this difference could be related to the association with regulatory subunits. As first step, we investigated the role of BKCa β1 subunit by characterizing cardiac mitoBKCa properties in wild type (wt) and β1 KO mice. In wt mice, mitoBKCa was frequently recorded (in 6 out of 7 patches and 3 different preparations), had a conductance of ∼313 ± 8 pS (n=4) resembling that of mitoBKCa channel from rat (∼305 ± 19 pS, n=7), and was Ca2+ (n=2) and paxilline sensitive (n=3). In contrast to rat, wt mice had predominantly one population of mitoBKCa with a high open probability (Po∼0.8) at all membrane potentials tested (−80 to +80 mV, in 12 µM Ca2+). Interestingly, while in wt mice mitoBKCa were frequently recorded, in β1 KO, mitoBKCa was recorded only in 5 out of 24 patches (n=5 preparations). This channel had a similar conductance as the wt channel but with a much lower Po (∼0.25 vs. ∼0.8 at +60 mV in 12 µM Ca2+). These results are consistent with the view that in cardiac mitochondria, mitoBKCa is forming a complex with its β1 subunit favoring mitoBKCa expression and a high channel activity. A contribution of alternative subunits to mitoBKCa targeting and function needs further investigation. Supported by NIH and AHA.

Effectors of large-conductance calcium-activated potassium channel modulate glutamate excitotoxicity in organotypic hippocampal slice cultures

Mitochondria have been suggested as a potential target for cytoprotective strategies. It has been shown that increased K+ uptake mediate by mitochondrial ATP-regulated potassium channels (mitoKATP channel) or large-conductance Ca2+-activated potassium channels (mitoBKCa channel) may provide protection in different models of cell death. Since recent findings demonstrated the presence of BKCa channels in neuronal mitochondria, the goal of the present study was to test the potential neuroprotective effects of BKCa channel modulators. Using organotypic hippocampal slice cultures exposed to glutamate, we demonstrated that preincubation of the slices with the BKCa channel opener NS1619 resulted in decreased neuronal cell death measured as reduced uptake of propidium iodide. This neuroprotective effect was reversed by preincubation with the BKCa channel inhibitors paxilline and Iberiotoxin (IbTx). Moreover, mitochondrial respiration measurements revealed that NS1619 induced an IbTx-sensitive increase in state 2 respiration of isolated brain mitochondria. In addition, electrophysiological patch-clamp studies confirmed the presence of BKCa channels in mitoplasts isolated from embryonic hippocampal cells. Taken together, our results confirm presence of BKCa channel in rat hippocampal neurons mitochondria and suggest putative role for mitoBKCa in neuroprotection.

Carbon monoxide released by CORM-401 uncouples mitochondrial respiration and inhibits glycolysis in endothelial cells: A role for mitoBKCa channels

Carbon monoxide (CO), a product of heme degradation by heme oxygenases, plays an important role in vascular homeostasis. Recent evidence indicates that mitochondria are among a number of molecular targets that mediate the cellular actions of CO. In the present study we characterized the effects of CO released from CORM-401 on mitochondrial respiration and glycolysis in intact human endothelial cells using electron paramagnetic resonance (EPR) oximetry and the Seahorse XF technology. We found that CORM-401 (10–100μM) induced a persistent increase in the oxygen consumption rate (OCR) that was accompanied by inhibition of glycolysis (extracellular acidification rate, ECAR) and a decrease in ATP-turnover. Furthermore, CORM-401 increased proton leak, diminished mitochondrial reserve capacity and enhanced non-mitochondrial respiration. Inactive CORM-401 (iCORM-401) neither induced mitochondrial uncoupling nor inhibited glycolysis, supporting a direct role of CO in the endothelial metabolic response induced by CORM-401. Interestingly, blockade of mitochondrial large-conductance calcium-regulated potassium ion channels (mitoBKCa) with paxilline abolished the increase in OCR promoted by CORM-401 without affecting ECAR; patch-clamp experiments confirmed that CO derived from CORM-401 activated mitoBKCa channels present in mitochondria. Conversely, stabilization of glycolysis by MG132 prevented CORM-401-mediated decrease in ECAR but did not modify the OCR response. In summary, we demonstrated in intact endothelial cells that CO induces a two-component metabolic response: uncoupling of mitochondrial respiration dependent on the activation of mitoBKCa channels and inhibition of glycolysis independent of mitoBKCa channels.

Mitochondrial BKCa channel.

Since its discovery in a glioma cell line 15 years ago, mitochondrial BKCa channel (mitoBKCa) has been studied in brain cells and cardiomyocytes sharing general biophysical properties such as high K+ conductance (~300 pS), voltage-dependency and Ca2+-sensitivity. Main advances in deciphering the molecular composition of mitoBKCa have included establishing that it is encoded by the Kcnma1 gene, that a C-terminal splice insert confers mitoBKCa ability to be targeted to cardiac mitochondria, and evidence for its potential coassembly with β subunits. Notoriously, β1 subunit directly interacts with cytochrome c oxidase and mitoBKCa can be modulated by substrates of the respiratory chain. mitoBKCa channel has a central role in protecting the heart from ischemia, where pharmacological activation of the channel impacts the generation of reactive oxygen species and mitochondrial Ca2+ preventing cell death likely by impeding uncontrolled opening of the mitochondrial transition pore. Supporting this view, inhibition of mitoBKCa with Iberiotoxin, enhances cytochrome c release from glioma mitochondria. Many tantalizing questions remain open. Some of them are: how is mitoBKCa coupled to the respiratory chain? Does mitoBKCa play non-conduction roles in mitochondria physiology? Which are the functional partners of mitoBKCa? What are the roles of mitoBKCa in other cell types? Answers to these questions are essential to define the impact of mitoBKCa channel in mitochondria biology and disease.

Brain mitochondrial ATP-insensitive large conductance Ca+ 2-activated K+ channel properties are altered in a rat model of amyloid-β neurotoxicity

Mitochondrial dysfunction is a hallmark of amyloid-beta (Aβ)-induced neuronal toxicity in Alzheimer's disease (AD). However, the underlying mechanism of how Aβ affects mitochondrial function remains uncertain. Because mitochondrial potassium channels have been involved in several mitochondrial functions including cytoprotection, apoptosis and calcium homeostasis, a study was undertaken to investigate whether the gating behavior of the mitochondrial ATP- and ChTx-insensitive-IbTx-sensitive Ca2+-activated potassium channel (mitoBKCa) is altered in a rat model of Aβ neurotoxicity. Aβ1–42 (4μg/μl) was intracerebroventricularly injected in male Wistar rats (220–250g). Brain Aβ accumulation was confirmed two weeks later on the basis of an immunohistochemistry staining assay, and physiological impacts measured in passive avoidance task cognitive performance experiments. Brain mitochondrial inner membranes were then extracted and membrane vesicles prepared for channel incorporation into bilayer lipid. Purity of the cell fraction was confirmed by Western blot using specific markers of mitochondria, plasma membrane, endoplasmic reticulum, and Golgi.Our results first provide evidence for differences in mitoBKCa ion permeation properties with channels coming from Aβ vesicle preparations characterized by an inward rectifying I–V curve, in contrast to control mitoBKCa channels which showed a linear I–V relationship under the same ionic conditions (200mM cis/50mM trans). More importantly the open probability of channels from Aβ vesicles appeared 1.5 to 2.5 smaller compared to controls, the most significant decrease being observed at depolarizing potentials (30mV to 50mV). Because BKCa-β4 subunit has been documented to shift the BKCa channel voltage dependence curve, a Western blot analysis was undertaken where expression of mitoBKCa α and β4 subunits was estimated using anti-α and β4 subunit antibodies. Our results indicated a significant increase in mitoBKCa-β4 subunit expression coupled to a decrease in the expression of α subunit. Our results thus demonstrate a modification in the mitoBKCa channel gating properties in membrane preparations coming from a rat model of Aβ neurotoxicity, an effect potentially linked to a change in mitoBKCa-β4 and -α subunits expression or increased ROS production due to an enhanced Aβ mitochondrial accumulation. Our results may provide new insights into the cellular mechanisms underlying mitochondrial dysfunctions in Aβ neurotoxicity.

The Subproteome of mitoBKCa from Cardiomyocytes Reveals Novel Insights into BK Channel Function and Pathology

The mitochondrial large-conductance Ca2+ -activated K+ channel (mitoBKCa) of adult cardiomyocytes contains a C-terminal 50 amino acid insert (DEC) as essential for mitochondrial targeting. The activation of mitochondrial BK channels (mitoBKCa) protects the heart from ischemia and reperfusion injury, highlighting its significant role in cardiac function. Here, we characterized the subproteome of mitoBKCa in cardiomyocytes in the quest to better understand its physiological and pathological relevance in the heart. To this end, we used GST-DEC and GST proteins to pull down proteins interacting with DEC using cardiomyocyte lysates and mitochondrial lysates. We also used monoclonal BK antibody and IgG to immunoprecipitate proteins interacting with mitoBKCa using Percoll purified mitochondrial lysates from whole heart. In all, we identified 212 cellular proteins and 157 mitochondrial proteins interacting with DEC; and 60 mitochondrial proteins interacting with mitoBKCa. Gene oncology analysis revealed mitoBKCa potential contribution to multiple cellular functions. It could contribute to oxidative respiration through cytochrome c oxidase (5 subunits) and ADP/ATP translocase (AAC), including AAC1 (10 peptides, n=2) and AAC2 (13 peptides, n=2), and regulate mitochondrial metabolism through acetyl-Coenzyme transferase 2 (7 peptides, n=2). In addition, our data revealed that mitoBKCa has a broader impact. It might facilitate the pathological process of Parkinson's disease and multiple types of cancer by interacting with critical enzymes, such as NADH dehydrogenase (7 subunits, 3-7 peptides each) and integrin linked kinase (2 peptides, n =1). Excitingly, mitoBKCa interacted with 15 different subunits of the ATP synthase, which forms the permeability transition pore, underscoring a new mechanism for mitoBKCa to protect the heart. In summary, proteomic analysis identified a wide variety of cellular and mitochondrial proteins forming complex with mitoBKCa channel, revealing new paradigms for BK channel function.

Impairment of brain mitochondrial charybdotoxin- and ATP-insensitive BK channel activities in diabetes.

Existing evidence indicates an impairment of mitochondrial functions and alterations in potassium channel activities in diabetes. Because mitochondrial potassium channels have been involved in several mitochondrial functions including cytoprotection, apoptosis and calcium homeostasis, a study was carried out to consider whether the gating behavior of the mitochondrial ATP- and ChTx-insensitive Ca(2+)-activated potassium channel (mitoBKCa) is altered in a streptozotocin (STZ) model of diabetes. Using ion channel incorporation of brain mitochondrial inner membrane into the bilayer lipid membrane, we provide in this work evidence for modifications of the mitoBKCa ion permeation properties with channels from vesicles preparations coming from diabetic rats characterized by a significant decrease in conductance. More importantly, the open probability of channels from diabetic rats was reduced 1.5-2.5 fold compared to control, the most significant decrease being observed at depolarizing potentials. Because BKCa β4 subunit has been documented to left shift the BKCa channel voltage dependence curve in high Ca(2+) conditions, a Western blot analysis was undertaken where the expression of mitoBKCa α and β4 subunits was estimated using of anti-α and β4 subunit antibodies. Our results indicated a significant decrease in mitoBKCa β4 subunit expression coupled to a decrease in the expression of α subunit, an observation compatible with the observed decrease in Ca(2+) sensitivity. Our results thus demonstrate a modification in the mitoBKCa channel gating properties in membrane preparations coming from STZ model of diabetic rats, an effect potentially linked to a change in mitoBKCa β4 and α subunits expression and/or to an increase in reactive oxygen species production in high glucose conditions.

Putative Structural and Functional Coupling of the Mitochondrial BKCa Channel to the Respiratory Chain

Potassium channels have been found in the inner mitochondrial membranes of various cells. These channels regulate the mitochondrial membrane potential, the matrix volume and respiration. The activation of these channels is cytoprotective. In our study, the single-channel activity of a large-conductance Ca2+-regulated potassium channel (mitoBKCa channel) was measured by patch-clamping mitoplasts isolated from the human astrocytoma (glioblastoma) U-87 MG cell line. A potassium-selective current was recorded with a mean conductance of 290 pS in symmetrical 150 mM KCl solution. The channel was activated by Ca2+ at micromolar concentrations and by the potassium channel opener NS1619. The channel was inhibited by paxilline and iberiotoxin, known inhibitors of BKCa channels. Western blot analysis, immuno-gold electron microscopy, high-resolution immunofluorescence assays and polymerase chain reaction demonstrated the presence of the BKCa channel β4 subunit in the inner mitochondrial membrane of the human astrocytoma cells. We showed that substrates of the respiratory chain, such as NADH, succinate, and glutamate/malate, decrease the activity of the channel at positive voltages. This effect was abolished by rotenone, antimycin and cyanide, inhibitors of the respiratory chain. The putative interaction of the β4 subunit of mitoBKCa with cytochrome c oxidase was demonstrated using blue native electrophoresis. Our findings indicate possible structural and functional coupling of the mitoBKCa channel with the mitochondrial respiratory chain in human astrocytoma U-87 MG cells.

Coupling of the Electron Transport Chain with the Mitochondrial BKCa Channel in Rat Astrocytes

Potassium channels have been found in the inner mitochondrial membranes of various cells. These channels are regulating the mitochondrial membrane potential, the matrix volume and respiration. In our study, the single-channel activity of a large-conductance Ca2+-regulated potassium (mitoBKCa) channel was measured by a patch-clamping of mitoplasts isolated from rat astrocytes. A potassium-selective current was recorded with a mean conductance of 290 pS. The channel was inhibited by paxilline and iberiotoxin, inhibitors of BKCa channels. Western blot analysis and immunofluorescence demonstrated the presence of the BKCa channel β4 subunit in the inner mitochondrial membranes of the astrocytes. We have shown that substrates of the respiratory chain, such as NADH, succinate, and glutamate/malate are decreasing the activity of the channel at positive voltage. The effect was abolished by rotenone, antimycin and cyanide, inhibitors of the respiratory chain. The interaction of the β4 subunit of mitoBKCa with cytochrome c oxidase was demonstrated by using of Blue Native electrophoresis. Our findings indicate structural and functional coupling of an electron transport chain with the mitoBKCa channel in rat astrocytes.

Oxidized Heme - A Novel Inhibitor of Calcium-Dependent BK Channel in Rat Brain Mitochondria

Heme is a prosthetic group that consists of an iron atom bound in the center of a porphyrin ring. It is an essential element of hemeproteins in all living organisms. Although heme is ubiquitous, its circulation is strictly controlled. Therefore it is believed that it may play as yet unknown regulatory functions.The mitochondrial calcium-dependent BK channel (mitoBKCa) is one of the five known channels that contribute to potassium permeability of mitochondrial inner membrane. It is activated by calcium and voltage and inhibited by scorpion venom toxins such as charybdotoxin and iberiotoxin.In the current study, we have checked the impact of the oxidized heme (hemin) on mitochondrial membrane potential and respiration rate of rat brain mitochondria. We have shown that hemin prevents the collapse of membrane potential that is normally caused by calcium-dependent BK channel openers (NS1619). A similar, though modest effect was observed in studies of oxygen consumption rate. We also report inhibitory effects of hemin on the reactive oxygen species-downregulating protperties of NS1619.Additionally, we have studied the single channel activity of mitoBKCa by patch-clamp of mitoplasts isolated from a rat astrocyte cell line. The results that we have obtained confirm the phenomonon of reversible inhibition of mitoBKCa channel by hemin.Our findings support the hypothesis that oxidized heme can inhibit the mitochonbdrial calcium-dependent BK channel.This study is supported by the International PhD Programme (MPD/2009/4/styp16) coordinated by The Foundation for Polish Science and financed from the European Funds, it is also supported by the Polish Mitochondrial Network MitoNet.pl.

How many types of large conductance Ca+2-activated potassium channels exist in brain mitochondrial inner membrane: evidence for a new mitochondrial large conductance Ca2+-activated potassium channel in brain mitochondria

Recently, we have reported electropharmacological properties of a charybdotoxin (ChTx)- and ATP-insensitive-iberiotoxin (IbTx)-sensitive large conductance Ca⁺²-activated potassium (BKCa) channel in almost purified brain mitochondrial inner membrane vesicles. In this work, we report the single-channel characterization of a new BK channel from rat brain mitochondrial inner membrane (mitochondrial large conductance Ca²⁺-activated potassium channel, mitoBKCa channel) incorporated into a planar lipid bilayer. The channel conductance was 565 pS in 200 mM KCl cis/50 mM KCl trans. The channel open probability appeared voltage-independent at -40 to +40 mV. Adding 10 mM 4-aminopyridine (4-AP) at positive and negative potentials and 2.5 mM ATP at positive voltages inhibited the channel activities. Notably, addition of 70 μM glibenclamide to the cis side had no effect on the channel behavior. Hence, it can be concluded that there are, at least, two different types of mitoBKCa channels in brain mitochondrial membrane, and the IbTx-, ChTx-, and ATP-sensitive mitoBKCa channels may be activated during the decline of cell metabolism.